In the United States and throughout the world, cancer incidence and mortality has increased dramatically in both developed and developing nations. Cancer causes ~13% of human deaths with 7.6 million people dying from cancer in 2007. More people in the US die of lung cancer than breast, colon, kidney, and prostate cancers combined. Recent studies show that veterans are 25 to 75 percent more likely to develop lung cancer than people who did not serve in the military; yet therapies for lung cancer and other solid tumors are still limited. Early in the 1900's, Coley successfully used a mix of heat-killed Streptococcus and Serratia bacteria to treat a variety of sarcomas and other cancers. Similar treatments today with live BCG help prevent recurrence of bladder cancer. The effectiveness of these therapies provides evidence that it is possible to treat malignancies by activating the immune system with bacteria. A wide variety of approaches are now being tried to stimulate tumor immunity by 12 T cells using tumor-specific peptide antigens. However, since CD4 and CD8 12 T cells recognize these peptides bound to MHC molecules in an MHC-restricted fashion, immunotherapy with 12 T cells needs to be individualized for each MHC haplotype. In contrast, 34 T cells represent a unique subset of T cells that bridge innate and adaptive immunity by recognizing nonpeptide antigens in an MHC-unrestricted manner. The major subset of human 34 T cells use their V32V42 T cell receptors to recognize the foreign-microbial isoprenoid metabolite, HMBPP and the self- metabolite, IPP. V32V42 T cells expand in the blood with a variety of infections and then accumulate at peripheral sites. Recognition of HMBPP activates 34 T cells to produce Th1 cytokines, kill infected cells, and produce growth factors to repair mucosal surfaces. Human 34 T cells also help to regulate malignancies. Once activated, V32V42 T cells use their TCR and NK receptors to recognize and kill a wide variety of tumor cells irrespective of their tissue origin, MHC expression, or MHC-haplotype. Since V32V42 T cells also express the CD16 immunoglobulin Fc receptor, they kill tumor cells sensitized by anti-tumor antibodies. In recent years, immunotherapy with prenyl pyrophosphates or bisphosphonates and IL-2 to stimulate V32V42 T cells alone or in conjunction with rituximab for lymphoma has shown promise since these treatments resulted in complete and partial remissions or stable disease in patients with lymphoma and metastatic renal or prostate cancer. However, repeat immunizations lead to anergy and deletion of V32V42 T cells. Metabolic engineering of bacteria is a new field of study that has, up to now, been focused on altering bacteria for drug or chemical synthesis or for the generation of alternative fuels. Directed changes in bacterial metabolism are made by modifying specific biochemical reactions or by introducing new ones. No group has attempted to metabolically engineer bacteria to derive 34 vaccines. We now propose to develop new vaccines for V32V42 T cell cancer immunotherapy by metabolic engineering Salmonella and Listeria bacteria to overproduce HMBPP. Both species have been used for cancer vaccines but differ significantly since the Gram negative Salmonella uses the MEP pathway and is given orally whereas the Gram positive Listeria uses both the MEP and mevalonate pathways and is given intravenously. These fundamental differences may result in qualitatively different V32V42 stimulation so both will be pursued. These vaccines will activate V32V42 T cells to kill tumor cells by TCR and NKR recognition or by antibody-dependent cellular cytotoxicity through anti-tumor mAbs bound to CD16. To accomplish our goals, we will: (1) metabolically engineer vaccine bacteria to overproduce HMBPP, (2) test engineered bacteria in vitro for HMBPP levels, growth rates, virulence, and ability to infect and proliferate in human cells, (3) test engineered bacteria in vivo in monkeys, and (4) assess the ability of V32V42 T cells stimulated by metabolically engineered bacteria to control tumors. PUBLIC HEALTH RELEVANCE: Many studies have found higher rates of cancer incidence and mortality among veterans. The average age of a veteran is 58 years old with most between 45 to 64. In the United States, cancer accounts for 25% of all deaths with 30% of these from lung cancer. U.S. veterans who served in the Vietnam, Korean, or the 1991 Persian Gulf war are at high-risk for lung cancer since they are 25 to 75 percent more likely to develop lung cancer than people who did not serve. Also, veterans were exposed to various environmental hazards and microbial infections. Despite advances in our understanding of the causes of cancer, progress in treatment is still limited for many types of cancers. Our vaccine will stimulate a type of blood cells called gamma delta T cells for treatment of a number of different tumor types by immunotherapy.